Use of hmgb polypetides for increasing immune responses

ABSTRACT

The present invention features polypeptides comprising an HMGB B box or a functional variant thereof that are useful for stimulating or increasing an immune response in an individual. Such polypeptides can be used in vaccine formulations and in cancer therapies.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/427,848, filed Nov. 20, 2002. The entire teachings of the aboveapplication are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant RO1 GM57226 from the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

The immune system functions to destroy or neutralize foreign matter.Immune responses protect against infection by microbes, includingviruses, bacteria, fungi, and other parasites. In addition, the immunesystem functions to destroy the body's own cells that have becomeabnormal, for example, cancer cells, and cells that are old and nolonger useful to the body, such as erythrocytes.

Manipulation of the immune system is one way in which a therapeutic orprotective immune response can be mounted, and a number of diseases canbe treated through manipulation of the immune system. Current therapiesfor treating immunological disorders include anti-inflammatory agents,for example, corticosteroids, cytotoxic agents, agents that modulatesignaling events within the immune system, and antibodies.

There are many diseases in which the action of the immune system isinadequate. Therefore, there is a need for treatments of these diseases.

SUMMARY OF THE INVENTION

It has been found that HMGB polypeptides, as well as polypeptidescomprising an HMGB B box or a functional variant thereof (collectivelytermed “HMGB B boxes”) are useful for stimulating cytokine activity fromcells administered such polypeptides. Thus, HMGB polypeptides andpolypeptides comprising an HMGB B box can be used to increase an immuneresponse in an individual and to treat a number of diseases for which anincreased immune response is desired. Examples of conditions that can betreated using the reagents and methods as described herein includecancer and viral infections, including HIV/AIDS, allergic disease, andasthma. HMGB B boxes and functional variants described herein can alsobe used as part of a vaccine, in which an immune response is desired toprevent, ameliorate, or treat an infectious disease.

Accordingly, in one aspect, the invention features a pharmaceuticalcomposition comprising an HMGB polypeptide or a functional fragment orvariant thereof (collectively termed “HMGB polypeptides”), or an HMGB Bbox or a functional variant thereof (collectively termed “HMGB Bboxes”), in an amount sufficient to treat a disease or condition inwhich an increase in an immune response in an individual administeredthe pharmaceutical composition is desired. In one embodiment, thepharmaceutical composition further comprises a vaccine.

In another aspect, the invention features an antibody attached to apolypeptide comprising an HMGB polypeptide or a functional fragment orvariant thereof or an HMGB B box or a functional variant thereof. In oneembodiment, the antibody is in a pharmaceutically acceptable carrier.

In another embodiment, the invention features a method of stimulating orincreasing an immune response in an individual in need ofimmunostimulation, the method comprising administering to the individuala polypeptide comprising an HMGB polypeptide or a functional fragment orvariant thereof or an HMGB B box or a functional variant thereof. In oneembodiment, the individual is being treated for cancer. In anotherembodiment, the polypeptide is attached to an antibody specific to atarget site in the individual in need of immunostimulation. In anotherembodiment, the polypeptide is co-administered with a vaccine. Inanother embodiment, the polypeptide is in a pharmaceutically acceptablecarrier.

In another aspect, the invention features a method of treating cancer inan individual, the method comprising administering to the individual atherapeutically effective amount of a polypeptide comprising an HMGBpolypeptide or a functional fragment or variant thereof or an HMGB B boxor a functional variant thereof. In one embodiment, the individual isbeing treated for cancer. In another embodiment the polypeptide isattached to an antibody specific to a target site in the individual inneed of immunostimulation. In another embodiment, the polypeptide isco-administered with a vaccine. In another embodiment, the polypeptideis in a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of HMG1 mutants and their activityin TNF release (pg/ml).

FIG. 2A is a histogram showing the effect of 0 μg/ml, 0.01 μg/ml, 0.1μg/ml, 1 μg/ml or 10 μg/ml of B box on TNF release (pg/ml) in RAW 264.7cells.

FIG. 2B is a histogram showing the effect of 0 μg/ml, 0.01 μg/ml, 0.1μg/ml, 1 μg/ml or 10 μg/ml of B box on IL-1β release (pg/ml) in RAW264.7 cells.

FIG. 2C is a histogram showing the effect of 0 μg/ml, 0.01 μg/ml, 0.1μg/ml, 1 μg/ml or 10 μg/ml of B box on IL-6 release (pg/ml) in RAW 264.7cells.

FIG. 2D a scanned image of a blot of an RNAse protection assay, showingthe effect of B box (at 0 hours, 4 hours, 8 hours, or 24 hours afteradministration) or vector alone (at 4 hours after administration) on TNFmRNA expression in RAW 264.7 cells.

FIG. 2E is a histogram of the effect of HMG1 B box on TNF proteinrelease (pg/ml) from RAW 264.7 cells at 0 hours, 4 hours, 8 hours, 24hours, 32 hours or 48 hours after administration.

FIG. 2F is a histogram of the effect of vector on TNF protein release(pg/ml) from RAW 264.7 cells at 0 hours, 4 hours, 8 hours, 24 hours, 32hours or 48 hours after administration.

FIG. 3 is a schematic representation of HMG1 B box mutants and theiractivity in TNF release (pg/ml).

FIG. 4A is a scanned image of a hematoxylin and eosin stained kidneysection obtained from an untreated mouse.

FIG. 4B is a scanned image of a hematoxylin and eosin stained kidneysection obtained from a mouse administered HMG1 B box.

FIG. 4C is a scanned image of a hematoxylin and eosin stained myocardiumsection obtained from an untreated mouse.

FIG. 4D is a scanned image of a hematoxylin and eosin stained myocardiumsection obtained from a mouse administered HMG1 B box.

FIG. 4E is a scanned image of a hematoxylin and eosin stained lungsection obtained from an untreated mouse.

FIG. 4F is a scanned image of a hematoxylin and eosin stained lungsection obtained from a mouse administered HMG1 B box.

FIG. 4G is a scanned image of a hematoxylin and eosin stained liversection obtained from an untreated mouse.

FIG. 4H is a scanned image of a hematoxylin and eosin stained liversection obtained from a mouse administered HMG1 B box.

FIG. 4I is a scanned image of a hematoxylin and eosin stained liversection (high magnification) obtained from an untreated mouse.

FIG. 4J is a scanned image of a hematoxylin and eosin stained liversection (high magnification) obtained from a mouse administered HMG Bbox.

FIG. 5A is the amino acid sequence of a human HMG1 polypeptide (SEQ IDNO: 1).

FIG. 5B is the amino acid sequence of rat and mouse HMG1 (SEQ ID NO: 2).

FIG. 5C is the amino acid sequence of human HMG2 (SEQ ID NO: 3).

FIG. 5D is the amino acid sequence of a human, mouse, and rat HMG1 A boxpolypeptide (SEQ ID NO: 4).

FIG. 5E is the amino acid sequence of a human, mouse, and rat HMG1 B boxpolypeptide (SEQ ID NO: 5).

FIG. 5F is the nucleic acid sequence of a forward primer for human HMG1(SEQ ID NO: 6).

FIG. 5G is the nucleic acid sequence of a reverse primer for human HMG1(SEQ ID NO: 7).

FIG. 5H is the nucleic acid sequence of a forward primer for the carboxyterminus mutant of human HMG1 (SEQ ID NO: 8).

FIG. 5I is the nucleic acid sequence of a reverse primer for the carboxyterminus mutant of human HMG1 (SEQ ID NO: 9).

FIG. 5J is the nucleic acid sequence of a forward primer for the aminoterminus plus B box mutant of human HMG1 (SEQ ID NO: 10).

FIG. 5K is the nucleic acid sequence of a reverse primer for the aminoterminus plus B box mutant of human HMG1 (SEQ ID NO: 11).

FIG. 5L is the nucleic acid sequence of a forward primer for a B boxmutant of human HMG1 (SEQ ID NO: 12).

FIG. 5M is the nucleic acid sequence of a reverse primer for a B boxmutant of human HMG1 (SEQ ID NO: 13).

FIG. 5N is the nucleic acid sequence of a forward primer for the aminoterminus plus A box mutant of human HMG1 (SEQ ID NO: 14).

FIG. 5O is the nucleic acid sequence of a reverse primer for the aminoterminus plus A box mutant of human HMG1 (SEQ ID NO: 15).

FIG. 6 is a sequence alignment of HMG1 polypeptide sequence from rat(SEQ ID NO:2), mouse (SEQ ID NO:2), and human (SEQ ID NO: 18).

FIG. 7A is the nucleic acid sequence of HMG1L5 (formerly HMG1L10) (SEQID NO: 32) encoding an HMGB polypeptide.

FIG. 7B is the polypeptide sequence of HMG1L5 (formerly HMG1L10) (SEQ IDNO: 24) encoding an HMGB polypeptide.

FIG. 7C is the nucleic acid sequence of HMG1L1 (SEQ ID NO: 33) encodingan HMGB polypeptide.

FIG. 7D is the polypeptide sequence of HMG1L1 (SEQ ID NO: 25) encodingan HMGB polypeptide.

FIG. 7E is the nucleic acid sequence of HMG1L4 (SEQ ID NO: 34) encodingan HMGB polypeptide.

FIG. 7F is the polypeptide sequence of HMG1L4 (SEQ ID NO: 26) encodingan HMGB polypeptide.

FIG. 7G is the nucleic acid sequence of the HMG polypeptide sequence ofthe BAC clone RP11-395A23 (SEQ ID NO: 35).

FIG. 7H is the polypeptide sequence of the HMG polypeptide sequence ofthe BAC clone RP11-395A23 (SEQ ID NO: 27) encoding an HMGB polypeptide.

FIG. 7I is the nucleic acid sequence of HMG1L9 (SEQ ID NO: 36) encodingan HMGB polypeptide.

FIG. 7J is the polypeptide sequence of HMG1L9 (SEQ ID NO: 28) encodingan HMGB polypeptide.

FIG. 7K is the nucleic acid sequence of LOC122441 (SEQ ID NO: 37)encoding an HMGB polypeptide.

FIG. 7L is the polypeptide sequence of LOC122441 (SEQ ID NO: 29)encoding an HMGB polypeptide.

FIG. 7M is the nucleic acid sequence of LOC139603 (SEQ ED NO: 38)encoding an HMGB polypeptide.

FIG. 7N is the polypeptide sequence of LOC139603 (SEQ ID NO: 30)encoding an HMGB polypeptide.

FIG. 7O is the nucleic acid sequence of HMG1L8 (SEQ ID NO: 39) encodingan HMGB polypeptide.

FIG. 7P is the polypeptide sequence of HMG1L8 (SEQ ID NO: 31) encodingan HMGB polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features HMGB polypeptides and polypeptidescomprising an HMGB B box or a functional variant thereof that are usefulfor stimulating or increasing an immune response in an individual. Inone embodiment the polypeptide comprises or consists of a mammalian HMGBB box, for example, a human HMGB B box. Examples of an HMGB B boxesinclude polypeptides having the sequence of SEQ ID NO: 5, SEQ ID NO: 20,or SEQ ID NO: 45.

As used herein, an “HMGB polypeptide” or an “HMGB protein” is anisolated, substantially pure, or substantially pure and isolatedpolypeptide that has been separated from components that naturallyaccompany it or a recombinantly produced polypeptide having the sameamino acid sequence, and increases inflammation, and/or increasesrelease of a proinflammatory cytokine from a cell, and/or increases theactivity of the inflammatory cytokine cascade. In one embodiment, theHMGB polypeptide has one of the above biological activities. In anotherembodiment, the HMGB polypeptide has two of the above biologicalactivities. In a third embodiment, the HMGB polypeptide has all three ofthe above biological activities.

Preferably, the HMGB polypeptide is a mammalian HMGB polypeptide, forexample, a human HMGB1 polypeptide. Examples of an HMGB polypeptideinclude a polypeptide comprising or consisting of the sequence of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 18. Preferably, theHMGB polypeptide contains a B box DNA binding domain and/or an A box DNAbinding domain, and/or an acidic carboxyl terminus as described herein.Other examples of HMGB polypeptides are described in GenBank AccessionNumbers AAA64970, AAB08987, P07155, AAA20508, S29857, P09429, NP 002119,CAA31110, S02826, U00431, X67668, NP_(—)005333, NM_(—)016957, andJ04179, the entire teachings of which are incorporated herein byreference. Additional examples of HMGB polypeptides include, but are notlimited to mammalian HMG1 ((HMGB1) as described, for example, in GenBankAccession Number U51677), HMG2 ((HGB2) as described, for example, inGenBank Accession Number M83665), HMG-2A ((HMGB3, HMG-4) as described,for example, in GenBank Accession Numbers NM_(—)005342 andNP_(—)005333), HMG14 (as described, for example, in GenBank AccessionNumber P05114), HMG17 (as described, for example, in GenBank AccessionNumber X13546), HMGI (as described, for example, in GenBank AccessionNumber L17131), and HMGY (as described, for example, in GenBankAccession Number M23618); nonmammalian HMG T1 (as described, forexample, in GenBank Accession Number X02666) and HMG T2 (as described,for example, in GenBank Accession Number L32859) (rainbow trout); HMG-X(as described, for example, in GenBank Accession Number D30765)(Xenopus); HMG D (as described, for example, in GenBank Accession NumberX71138) and IMG Z (as described, for example, in GenBank AccessionNumber X71139) (Drosophila); NHP10 protein (HMG protein homolog NIP 1)(as described, for example, in GenBank Accession Number Z48008) (yeast);non-histone chromosomal protein (as described, for example, in GenBankAccession Number 000479) (yeast); HMG 1/2 like protein (as described,for example, in GenBank Accession Number Z11540) (wheat, maize,soybean); upstream binding factor (UBF-1) (as described, for example, inGenBank Accession Number X53390); PMS1 protein homolog 1 (as described,for example, in GenBank Accession Number U13695); single-strandrecognition protein (SSRP, structure-specific recognition protein) (asdescribed, for example, in GenBank Accession Number M86737); the HMGhomolog TDP-1 (as described, for example, in GenBank Accession NumberM74017); mammalian sex-determining region Y protein (SRY,testis-determining factor) (as described, for example, in GenBankAccession Number X53772); fungal proteins: mat-1 (as described, forexample, in GenBank Accession Number AB009451), ste 11 (as described,for example, in GenBank Accession Number x53431) and Mc 1; SOX 14 (asdescribed, for example, in GenBank Accession Number AF107043) (as wellas SOX 1 (as described, for example, in GenBank Accession NumberY13436), SOX 2 (as described, for example, in GenBank Accession NumberZ31560), SOX 3 (as described, for example, in GenBank Accession NumberX71135), SOX 6 (as described, for example, in GenBank Accession NumberAF309034), SOX 8 (as described, for example, in GenBank Accession NumberAF226675), SOX 10 (as described, for example, in GenBank AccessionNumber AJ001183), SOX 12 (as described, for example, in GenBankAccession Number X73039) and SOX 21 (as described, for example, inGenBank Accession Number AF107044)); lymphoid specific factor (LEF-1)(asdescribed, for example, in GenBank Accession Number X58636); T-cellspecific transcription factor (TCF-1)(as described, for example, inGenBank Accession Number X59869); MTT1 (as described, for example, inGenBank Accession Number M62810); and SP100-HMG nuclear autoantigen (asdescribed, for example, in GenBank Accession Number U36501).

Other examples of HMGB proteins are polypeptides encoded by HMGB nucleicacid sequences having GenBank Accession Numbers NG_(—)000897 (HMG1L5(formerly HMG1L10)) (and in particular by nucleotides 150-797 ofNG_(—)000897, as shown in FIGS. 7A and 7B); AF076674 (HMG1L1) (and inparticular by nucleotides 1-633 of AF076674, as shown in FIGS. 7C and7D; AF076676 (HMG1L4) (and in particular by nucleotides 1-564 ofAF076676, as shown in FIGS. 7E and 7F); AC010149 (HMG sequence from BACclone RP11-395A23) (and in particular by nucleotides 75503-76117 ofAC010149), as shown in FIGS. 7G and 7H); AF165168 (HMG1L9) (and inparticular by nucleotides 729-968 of AF165168, as shown in FIGS. 7I and7J); XM_(—)063129 (LOC122441) (and in particular by nucleotides 319-558of XM_(—)063129, as shown in FIGS. 7K and 7L); XM_(—)066789 (LOC139603)(and in particular by nucleotides 1-258 of XM_(—)066789, as shown inFIGS. 7M and 7N); and AF165167 (HMG1L8) (and in particular bynucleotides 456-666 of AF165167, as shown in FIGS. 70 and 7P).

The HMGB polypeptides of the present invention also encompass sequencevariants. Variants include a substantially homologous polypeptideencoded by the same genetic locus in an organism, i.e., an allelicvariant, as well as other variants. Variants also encompass polypeptidesderived from other genetic loci in an organism, but having substantialhomology to a polypeptide encoded by an HMGB nucleic acid molecule, andcomplements and portions thereof or having substantial homology to apolypeptide encoded by a nucleic acid molecule comprising the nucleotidesequence of an HMGB nucleic acid molecule. Examples of HMGB nucleic acidmolecules are known in the art and can be derived from HMGB polypeptidesas described herein. Variants also include polypeptides substantiallyhomologous or identical to these polypeptides but derived from anotherorganism, i.e., an ortholog. Variants also include polypeptides that aresubstantially homologous or identical to these polypeptides that areproduced by chemical synthesis. Variants also include polypeptides thatare substantially homologous or identical to these polypeptides that areproduced by recombinant methods. Preferably, the HMGB polypeptide has atleast 60%, more preferably, at least 70%, 75%, 80%, 85%, or 90%, andmost preferably at least 95% sequence identity to a sequence selectedfrom SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:18, asdetermined using the BLAST program and parameters described herein andone of more of the biological activities of an HMGB polypeptide(functional variant).

In other embodiments, the present invention is directed to an HMGBpolypeptide fragment that has HMGB biological activity (functionalfragment). By an “HMGB polypeptide fragment that has HMGB biologicalactivity” or a “biologically active HMGB fragment” is meant a fragmentof an HMGB polypeptide that has the activity of an HMGB polypeptide. Anexample of such an HMGB polypeptide fragment is the HMGB B box, asdescribed herein. Biologically active HMGB fragments can be generatedusing standard molecular biology techniques and assaying the function ofthe fragment by determining if the fragment, when administered to a cellincrease release of a proinflammatory cytokine from the cell, comparedto a suitable control, for example, using methods described herein.

As used herein, an “HMGB B box” also referred to herein as a “B box” isa substantially pure, or substantially pure and isolated polypeptidethat has been separated from components that naturally accompany it, andconsists of an amino acid sequence that is less than a full length HMGBpolypeptide and has one or more of the following biological activities:increasing inflammation, increasing release of a proinflammatorycytokine from a cell, and/or increasing the activity of the inflammatorycytokine cascade. In one embodiment, the HMGB B box polypeptide has oneof the above biological activities. In another embodiment, the HMGB Bbox polypeptide has two of the above biological activities. In a thirdembodiment, the HMGB B box polypeptide has all three of the abovebiological activities. Preferably, the HMGB B box has at least 25%, 30%,40%, 50%, 60%, 70%, 80% or 90% of the biological activity of full lengthHMG. In another embodiment, the HMGB B box does not comprise an HMGB Abox. In another embodiment, the HMGB B box is a fragment of and HMGBpolypeptide (i.e., a polypeptide that is about 90%, 80%, 70%, 60%, 50%,40%, 35%, 30%, 25%, or 20% the length of a full length HMG1polypeptide). In another embodiment, the HMGB B box comprises orconsists of the sequence of SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 45,or the amino acid sequence in the corresponding region of an HMGBprotein in a mammal, but is still less than the full length HMGBpolypeptide. An HMGB B box polypeptide is also a recombinantly producedpolypeptide having the same amino acid sequence as an HMGB B boxpolypeptide described above. Preferably, the HMGB B box is a mammalianHMGB B box, for example, a human HMGB1 B box. An HMGB B box often has nomore than about 85 amino acids and no fewer than about 4 amino acids.

Examples of polypeptides having B box sequences within them include, butare not limited to HMGB polypeptides described herein. The B boxsequences in such polypeptides can be determined and isolated usingmethods described herein, for example, by sequence comparisons to Bboxes described herein and testing for B box biological activity. Inparticularly preferred embodiments, the B box comprises SEQ ID NO:5, SEQED NO:20, or SEQ ID NO:45, which are the sequences (three differentlengths) of the human HMGB1 B box, or is a fragment of an HMGB B boxthat has B box biological activity. For example, a 20 amino acidsequence contained within SEQ ID NO:20 contributes to the function ofthe B box. This 20 amino acid B-box fragment has the following aminoacid sequence: fkdpnapkrl psafflfcse (SEQ ID NO:23). Another example ofan HMGB B box biologically active fragment consists of amino acids 1-20of SEQ ID NO:5 (napkrppsaf flfcseyrpk; SEQ ID NO:16).

Examples of HMGB B box polypeptide sequences include the followingsequences: FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVAKK LGEMWNNTAADDKQPYEKKA AKLKEKYEKD IAAY (human HMGB1; SEQ ID NO: 17); KKDPNAPKRPPSAFFLFCSE HRPKIKSEHP GLSIGDTAKK LGEMWSEQSA KDKQPYEQKA AKLKEKYEKD IAAY(human HMGB2; SEQ ID NO: 40); FKDPNAPKRL PSAFFLFCSE YRPKIKGEHPGLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY (HMG1L5 (formerlyHMG1L10); SEQ ID NO: 41); FKDPNAPKRP PSAFFLFCSE YHPKIKGEHP GLSIGDVAKKLGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY (HMG1L1; SEQ ID NO: 42);FKDSNAPKRP PSAFLLFCSE YCPKIKGEHP GLPISDVAKK LVEMWNNTFA DDKQLCEKKAAKLKEKYKKD TATY (HMG1L4; SEQ ID NO: 43); FKDPNAPKRP PSAFFLFCSEYRPKIKGEHP GLSIGDVAKK LAGMWNNTAA ADKQFYEKKA AKLKEKYKKD IAAY (HMGsequence from BAC clone RP11-359A23; SEQ ID NO: 44); and FKDPNAPKRPPSAFFLFCSE YRPKIKGEHP GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKDIAAYRAKGKP DAAKKGVVKA EK (human HMGB1 box; SEQ ID NO: 45).

The HMGB B box polypeptides of the invention also encompasses sequencevariants that are functional variants, and can be naturally-occurring ornon-naturally-occurring. Functional variants include a substantiallyhomologous polypeptide encoded by the same genetic locus in an organism,i.e., an allelic variant, as well as other variants. Functional variantsalso encompass polypeptides derived from other genetic loci in anorganism, but having substantial homology to a polypeptide encoded by anHMGB nucleic acid molecule, and complements and portions thereof, orhaving substantial homology to a polypeptide encoded by a nucleic acidmolecule comprising the nucleotide sequence of an HMGB B box nucleicacid molecule. Examples of HMGB B box nucleic acid molecules are knownin the art and can be derived from HMGB B box polypeptides as describedherein. Functional variants also include polypeptides substantiallyhomologous or identical to these polypeptides but derived from anotherorganism, i.e., an ortholog. Functional variants also includepolypeptides that are substantially homologous or identical to thesepolypeptides that are produced by chemical synthesis. Functionalvariants also include polypeptides that are substantially homologous oridentical to these polypeptides that are produced by recombinantmethods:

Preferably, an HMGB B box polypeptide variant has at least 60%, morepreferably, at least 70%, 75%, 80%, 85%, or 90%, and most preferably atleast 95% sequence identity to the sequence of an HMGB B box asdescribed herein, for example, the sequence of SEQ ID NO: 5, SEQ ID NO:20, or SEQ ID NO: 45, as determined using the BLAST program andparameters described herein. Preferably, the HMGB B box consists of thesequence of SEQ ID NO: 5, SEQ ID NO: 20, or SEQ ID NO: 45, or the aminoacid sequence in the corresponding region of an HMGB protein in amammal, and has one or more of the biological activities of an HMGB Bbox, determined using methods described herein or other methods known inthe art.

As used herein, two polypeptides (or a region of the polypeptides) aresubstantially homologous or identical when the amino acid sequences areat least about 60%, 70%, 75%, 80%, 85%, 90% or 95% or more homologous oridentical. The percent identity of two amino acid sequences (or twonucleic acid sequences) can be determined by aligning the sequences foroptimal comparison purposes (e.g., gaps can be introduced in thesequence of a first sequence). The amino acids or nucleotides atcorresponding positions are then compared, and the percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., % identity=# of identicalpositions/total # of positions×100). In certain embodiments, the lengthof the HMGB polypeptide or HMGB B box polypeptide aligned for comparisonpurposes is at least 30%, preferably, at least 40%, more preferably, atleast 60%, and even more preferably, at least 70%, 80%, 90%, or 100% ofthe length of the reference sequence, for example, those sequenceprovided herein. The actual comparison of the two sequences can beaccomplished by well-known methods, for example, using a mathematicalalgorithm. A preferred, non-limiting example of such a mathematicalalgorithm is described in Karlin et al. (Proc. Natl. Acad. Sci. USA,90:5873-5877, 1993). Such an algorithm is incorporated into the BLASTNand BLASTX programs (version 2.2) as described in Schaffer et al.(Nucleic Acids Res., 29:2994-3005, 2001). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., BLASTN) can be used. In one embodiment, the database searched isa non-redundant (NR) database, and parameters for sequence comparisoncan be set at: no filters; Expect value of 10; Word Size of 3; theMatrix is BLOSUM62; and Gap Costs have an Existence of 11 and anExtension of 1.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0), which is part of the GCG (Accelrys, San Diego,Calif.) sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Additional algorithms for sequence analysis are known in the art andinclude ADVANCE and ADAM as described in Torellis and Robotti, Comput.Appl. Biosci., 10: 3-5, 1994; and FASTA described in Pearson and Lipman,Proc. Natl. Acad. Sci USA, 85: 2444-2448, 1988.

In another embodiment, the percent identity between two amino acidsequences can be accomplished using the GAP program in the GCG softwarepackage (Accelerys) using either a Blossom 63 matrix or a PAM250 matrix,and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or4. In yet another embodiment, the percent identity between two nucleicacid sequences can be accomplished using the GAP program in the GCGsoftware package (Accelrys), using a gap weight of 50 and a lengthweight of 3.

HMGB polypeptides of functional fragments or variants thereof(collectively termed “HMGB polypeptides”), or polypeptides comprising anHMGB B box or a functional variant thereof (collectively termed “HMGB Bboxes”) can be used in pharmaceutical compositions to stimulate orincrease an immune response. As used herein, by an “immune response” ismeant a collective and coordinated response to the introduction of aforeign substance in the body, by cells and molecules of the immunesystem. Cytokines play an important role in mediating immune responses.Thus molecules that stimulate cytokine activity are useful fordeveloping and/or mediating immune responses.

In one embodiment, the pharmaceutical composition comprises the HMGB Bbox and a vaccine. The vaccine can be administered to a person in needof immunostimulation (i.e., a person who would benefit by mounting orincreasing an immune response to an antigen, a tumor cell or a tumor) inorder to stimulate an immune response. Examples of vaccines includeHepatitis B Diptheria, Tetanus, Pertussis, Haemoplilus influenzae TypeB, Inactivated Polio, Measles, Mumps, Rubella, Varicella, Pneumococcal,Hepatitis A, Influenza, Japanese Encephalitis, Rotavirus, Yellow Fever,Trypanosoma cruzi; and Rabies. If desired, the pharmaceuticalcomposition can further comprise an adjuvant. As used herein, an“adjuvant” is an immunologic reagent that increases an antigenicresponse. Examples of adjuvants for use in pharmaceuticals includeimmunostimulatory oligonucleotides, imidazoquinolines (e.g., imiquimod),monophosphoryl lipid A, and detoxified lipopolysaccharide (LPS), asdescribed, for example, by O'Hagan et al. (Biomol. Eng. 18:69-85,2001)). An example of an immunostimulatory oligonucleotide is anoligonucleotide having ummethylated CpG sequences.

In another example, the pharmaceutical composition comprises an HMGBpolypeptide or functional fragment or variant thereof or an HMGB B boxpolypeptide or functional variant thereof attached to an antibody. Theantibody specifically binds a polypeptide, preferably an epitope, or atarget site (as determined, for example, by immunoassays, a techniquewell known in the art for assaying specific antibody-antigen binding) todeliver the HMGB B box polypeptide to the target site in order tostimulate or increase an immune response at the site where the antibodybinds. Antibodies of the invention include, but are not limited to,polyclonal, monoclonal, multispecific, human, humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′) fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, for example, anti-Id antibodies to antibodies ofthe invention), and epitope-binding fragments of any of the above.

The term “antibody,” as used herein, refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, andmore specifically, molecules that contain an antigen binding site thatspecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (for example, IgG, IgE, IgM, IgD, IgA andIgY), and of any class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2) or subclass of an immunoglobulin molecule.

In one embodiment, the antibodies are antigen-binding antibody fragmentsand include, without limitation, Fab, Fab′ and F(ab′)₂, Fd, single-chainFvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) andfragments comprising either a V_(L) or V_(H) domain. Antigen-bindingantibody fragments, including single-chain antibodies, can comprise thevariable region(s) alone or in combination with the entirety or aportion of one or more of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are antigen-binding fragmentsalso comprising any combination of variable region(s) with a hingeregion, CH1, CH2, and/or CH3 domains.

The antibodies of the invention may be from any animal origin includingbirds and mammals. Preferably, the antibodies are human, murine, donkey,sheep, rabbit, goat, guinea pig, hamster, horse, or chicken.

As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies producedby human B cells, or isolated from human sera, human immunoglobulinlibraries or from animals transgenic for one or more humanimmunoglobulins and that do not express endogenous immunoglobulins, asdescribed in U.S. Pat. No. 5,939,598 by Kucherlapati et al., forexample.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. The term “epitope,” as usedherein, refers to a portion of a polypeptide which contacts anantigen-binding site(s) of an antibody or T cell receptor.

The term “target site” as used herein, refers to a polypeptide that isrecognized by an antibody and to which the antibody binds. The targetsite is preferably a site at which delivery or localization of an HMGB Bbox polypeptide or functional variant thereof is desired. The targetsite can be in vivo or ex vitro. The target site can be, for example, apolypeptide localized on the surface of a cell or near (e.g., adjacentto) a cell to which delivery of an HMGB B box is desired. In oneembodiment the target site is a cancer target site, for example, acancer cell or a site near a tumor, such that delivery of an HMGBpolypeptide to the cancer cell or tumor occurs. In such a case, theantibody may be a tumor-associated antibody (i.e., an antibody that ispreferentially or exclusively bound by a cancer cell or tumor).

In one embodiment, the antibody of the present invention, attached to anHMGB B box or functional variant thereof is a tumor-associated antibodythat binds to a tumor-associated polypeptide, marker, or antigen at acancer target site. Tumor-associated polypeptides or markers include,but are not limited to oncofetal antigens, placental antigens, oncogenicor tumor virus-associated antigens, tissue-associated antigens,organ-associated antigens, ectopic hormones and normal antigens orvariants thereof. A sub-unit of a tumor-associated marker can also beused to raise antibodies having higher tumor-specificity, e.g., thebeta-subunit of human chorionic gonadotropin (HCG), which stimulates theproduction of antibodies having a greatly reduced cross-reactivity tonon-tumor substances. Suitable such marker substances to which specificantibodies may be raised and/or obtained which are useful in the presentinvention include, but are not limited to, alpha-fetoprotein (AFP),human chorionic gonadotropin (HCG) and/or its beta-subunit (HCG-beta),colon-specific antigen-p (CSAp), prostatic acid phosphatase, pancreaticoncofetal antigen, placental alkaline phosphatase, pregnancybeta₁-globulin, parathormone, calcitonin, tissue polypeptide antigen,T-antigen, beta₂-microglobulin, mammary tumor-associated glycoproteins(MTGP), galactyosyl transferase-II (GT-II), gp-52 viral-associatedantigen, ovarian cystadenocarcinoma-associated antigen (OCAA), ovariantumor-specific antigen (OCA), cervical cancer antigens (CA-58, CCA,TA-4), basic fetoprotein (BFP), terminal deoxynucleotidyl transferase(TdT), cytoplasmic melanoma-associated antigens, humanastrocytoma-associated antigen (HAAA), common glioma antigen (CGA),glioembryonic antigen (GEA), glial fibrillary acidic protein (GFA),common meningioma antigen (CMA), ferritin, and tumor angiogenesis factor(TAF).

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies used in the presentinvention may not display significant cross-reactivity, such that theydo not bind any other analog, ortholog, or homolog of a polypeptide ofthe present invention. Alternatively, antibodies of the invention canbind polypeptides with at least about 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, or 50% identity (as calculated using methods known in the art)to a polypeptide at a target site.

Antibodies of the present invention can also be described or specifiedin terms of their binding affinity to a polypeptide at a target site.Preferred binding affinities include those with a dissociation constantor Kd less than 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸M, 10⁻⁹M,5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰M, 5×10⁻¹⁰M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹³ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Antibodies used in the present invention can act as agonists orantagonists of a polypeptide at a target site. For example, the presentinvention includes antibodies which disrupt interactions with thepolypeptides at the target site either partially or fully. The inventionalso includes antibodies that do not prevent binding, but preventactivation or activity of the polypeptide. Activation or activity (forexample, signaling) may be determined by techniques known in the art.Also included are antibodies that prevent both binding to and activityof a polypeptide at a target site. Likewise included are neutralizingantibodies.

The antibodies used in the invention include derivatives that do notprevent the antibody from recognizing its epitope. For example, but notby way of limitation, the antibody derivatives include antibodies thathave been modified, for example, by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, or proteolytic cleavage.

The antibodies used in the invention can be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, or thelike, to induce the production of sera containing polyclonal antibodiesspecific for the antigen. Various adjuvants can be used to increase theimmunological response, depending on the host species, and include, butare not limited to, Freund's adjuvant (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesalso known in the art, including hybridoma cell culture, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniques as isknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).The term “monoclonal antibody” as used herein is not necessarily limitedto antibodies produced through hybridoma technology, but also refers toan antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone.

Human antibodies are desirable for therapeutic treatment of humanpatients. These antibodies can be made by a variety of methods known inthe art including phage display methods using antibody libraries derivedfrom human immunoglobulin sequences. Human antibodies can also beproduced using transgenic mice that are incapable of expressingfunctional endogenous immunoglobulins, but which can express humanimmunoglobulin genes. The transgenic mice are immunized with a selectedantigen, for example, all or a portion of a polypeptide of theinvention. Monoclonal antibodies directed against the antigen can beobtained from the immunized, transgenic mice using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, forexample, PCT publications WO 98/24893; WO 96/34096; WO 96/33735; andU.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; and 5,939,598.

An HMGB polypeptide or HMGB B box polypeptide can be attached, coupled,or conjugated to an antibody using methods known to one of skill in theart. In one embodiment, the polypeptide is covalently attached to theantibody. In another embodiment the polypeptide-antibody conjugate isproduced using recombinant methods, and is generated as a fusion proteincomprising the polypeptide and the antibody or an antigen bindingfragment of an antibody. Alternatively, the polypeptide can bechemically crosslinked to the antibody. If desired, spacers or linkers(for example, those available from Pierce Chemical Company) may be usedto attached the polypeptide to the linker. Methods for attaching apolypeptide to an antibody are described, for example, by Jeanson et al.(J. Immunol Methods 111:261-270, 1988); and Zarling et al. (Int. J.Immunopharmacol. 13 Suppl 1:63-68-1991). Reactive groups that can betargeted by coupling agents include primary amines, sulfhydryls, andcarbonyls.

The compositions of the invention can be administered alone or incombination with other therapeutic agents. Therapeutic agents that canbe administered in combination with the compositions of the invention,include but are not limited to chemotherapeutic agents, antibiotics,steroidal and non-steroidal anti-inflammatories, conventionalimmunotherapeutic agents, cytokines and/or growth factors. Combinationsmay be administered either concomitantly, for example, as an admixture,separately but simultaneously or concurrently; or sequentially.

In another embodiment, compositions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, taxol, andetoposide).

In an additional embodiment, the compositions of the invention may beadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha.

In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

As described herein, the compositions comprising HMGB polypeptides orfunctional fragments or variants thereof or HMGB B box polypeptides orfunctional variants thereof can be formulated in a pharmaceuticallyacceptable carrier. The pharmaceutically acceptable carrier includedwith the polypeptide in these compositions is chosen based on theexpected route of administration of the composition in therapeuticapplications. The route of administration of the composition depends onthe condition to be treated. For example, intravenous injection may bepreferred for treatment of a systemic disorder such as a leukemia orlymphoma, and oral administration may be preferred to treat agastrointestinal disorder such as a cancer of the gastrointestinalsystem, or an oral cancer. The route of administration and the dosage ofthe composition to be administered can be determined by the skilledartisan without undue experimentation in conjunction with standarddose-response studies. Relevant circumstances to be considered in makingthose determinations include the condition or conditions to be treated,the choice of composition to be administered, the age, weight, andresponse of the individual patient, and the severity of the patient'ssymptoms. Thus, depending on the condition, the composition can beadministered orally, parenterally, intranasally, vaginally, rectally,lingually, sublingually, bucally, intrabuccaly and transdermally to thepatient.

Accordingly, compositions designed for oral, lingual, sublingual, buccaland intrabuccal administration can be made without undue experimentationby means well known in the art, for example, with an inert diluent orwith an edible carrier. The compositions may be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the pharmaceutical compositions of the present inventionmay be incorporated with excipients and used in the form of tablets,troches, capsules, elixirs, suspensions, syrups, wafers, chewing gumsand the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth or gelatin. Examples of excipients includestarch or lactose. Some examples of disintegrating agents includealginic acid, corn starch and the like. Examples of lubricants includemagnesium stearate or potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring and the like. Materialsused in preparing these vanous compositions should be pharmaceuticallypure and non-toxic in the amounts used.

The compositions of the present invention can be administeredparenterally such as, for example, by intravenous, intramuscular,intrathecal or subcutaneous injection. Parenteral administration can beaccomplished by incorporating the antibody compositions of the presentinvention into a solution or suspension. Such solutions or suspensionsmay also include sterile diluents such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents. Parenteral formulations may also includeantibacterial agents such as, for example, benzyl alcohol or methylparabens, antioxidants such as, for example, ascorbic acid or sodiumbisulfite and chelating agents such as EDTA. Buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose may also be added. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

Rectal administration includes administering the pharmaceuticalcompositions into the rectum or large intestine. This can beaccomplished using suppositories or enemas. Suppository formulations caneasily be made by methods known in the art. For example, suppositoryformulations can be prepared by heating glycerin to about 120° C.,dissolving the antibody composition in the glycerin, mixing the heatedglycerin after which purified water may be added, and pouring the hotmixture into a suppository mold.

Transdermal administration includes percutaneous absorption of thecomposition through the skin. Transdermal formulations include patches,ointments, creams, gels, salves and the like.

The present invention includes nasally administering to the mammal atherapeutically effective amount of the composition. As used herein,nasally administering or nasal administration includes administering thecomposition to the mucous membranes of the nasal passage or nasal cavityof the patient. As used herein, pharmaceutical compositions for nasaladministration of a composition include therapeutically effectiveamounts of the agonist prepared by well-known methods to beadministered, for example, as a nasal spray, nasal drop, suspension,gel, ointment, cream or powder. Administration of the composition mayalso take place using a nasal tampon or nasal sponge.

Administration of the pharmaceutical compositions of the invention thepharmaceutical compositions of the invention can be administered toanimals, for example, humans in an amount sufficient to mount an immuneresponse for the treatment or prevention of a disease, for example aviral disease or a bacterial disease (e.g., through vaccination, orthrough anti-bacterial or anti-viral therapy), or to slow theproliferation of cancer cells or to kill them entirely, it would beclear to those skilled in the art that the optimal schedule foradministering such a pharmaceutical composition will vary based on thesubject, the subjects height and weight and the severity of the disease.Ultimately, the use and schedule of administration of a pharmaceuticalcomposition of the present invention will be decided by the treatingphysician, clinical protocols for determining dose range and schedulingare standard.

EXAMPLE 1 Materials and Methods

Cloning of HMGB1 and Production of HMGB1 B Box Mutants

The following methods were used to prepare clones and mutants of humanHMGB 1. Recombinant full length human HMGB 1 (651 base pairs; GenBankAccession Number U51677) was cloned by PCR amplification from a humanbrain Quick-Clone cDNA preparation (Clontech, Palo Alto, Calif.) usingthe following primers; forward primer: 5′ GATGGGCAAAGGAGATCCTAAG 3′ (SEQID NO: 6) and reverse primer: 5′ GCGGCCGCTTATTCATCATCATCATCTTC 3′ (SEQID NO: 7). Human HMGB1 mutants were cloned and purified as follows. Atruncated form of human HMGB 1 was cloned by PCR amplification from aHuman Brain Quick-Clone cDNA preparation (Clontech, Palo Alto, Calif.).The primers used were (forward and reverse, respectively): Carboxyterminus mutant (557 bp): (SEQ ID NO: 8) 5′ GATGGGCAAAGGAGATCCTAAG 3′and (SEQ ID NO: 9) 5′ GCGGCCGC TCACTTGCTTTTTTCAGCCTTGAC 3′. Aminoterminus + B box mutant (486 bp): (SEQ ID NO: 10)5′ GAGCATAAGAAGAAGCACCCA 3′ and (SEQ ID NO: 11) 5′ GCGGCCGCTCACTTGCTTTTTTCAGCCTTGAC 3′. B box mutant (233 bp): (SEQ ID NO: 12)5′ AAGTTCAAGGATCCCAATGCAAAG 3′ and (SEQ ID NO: 13)5′ GCGGCCGCTCAATATGCAGCTATATCCTTTTC 3′. Amino terminus + A box mutant(261 bp): (SEQ ID NO: 13) 5′ GATGGGCAAAGGAGATCCTAAG 3′ and (SEQ ID NO:14) 5′ TCACTTTTTTGTCTCCCCTTTGGG 3′.

A stop codon was added to each mutant to ensure the accuracy of proteinsize. PCR products were subcloned into pCRII-TOPO vector EcoRI sitesusing the TA cloning method per manufacturer's instruction (Invitrogen,Carlsbad, Calif.). After amplification, the PCR product was digestedwith EcoRI and subcloned onto expression vector with a GST tag pGEX(Pharmacia); correct orientation and positive clones were confirmed byDNA sequencing on both strands. The recombinant plasmids weretransformed into protease deficient E. Coli strains BL21 orBL21(DE3)plysS (Novagen, Madison, Wis.) and fusion protein expressionwas induced by isopropyl-D-thiogalactopyranoside (IPTG). Recombinantproteins were obtained using affinity purification with the glutathioneSepharose resin column (Pharmacia).

The HMGB mutants generated as described above have the following aminoacid sequences: Wild type HMGB 1: (SEQ ID NO: 18)MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEEDEEDEEDEEEE EDEEDEEDEEEDDDDECarboxy terminus mutant: (SEQ ID NO: 19)MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSK B Box mutant: (SEQ ID NO: 20)FKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAY Amino terminus + A Box mutant: (SEQ ID NO: 21)MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGET, wherein the A box consists of thesequence (SEQ ID NO: 22)PTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGET

A polypeptide generated from a GST vector lacking HMGB1 protein wasincluded as a control (containing a GST tag only). To inactive thebacterial DNA that bound to the wild type HMGB 1 and some of the mutants(carboxy terminus and B box), DNase I (Life Technologies), for carboxyterminus and B box mutants, or benzonase nuclease (Novagen, Madison,Wis.), for wild type HMGB1, was added at about 20 units/ml bacterialysate. Degradation of DNA was verified by ethidium bromide staining ofthe agarose gel containing HMGB1 proteins before and after thetreatment. The protein eluates were passed over a polymyxin B columnPierce, Rockford, Ill.) to remove any contaminating LPS, and dialyzedextensively against phosphate buffered saline to remove excess reducedglutathione. The preparations were then lyophilized and redissolved insterile water before use. LPS levels were less than 60 pg/μg protein forall the mutants and 300 pg/μg for wild type HMG-1 as measured by Limulusamebocyte lysate assay (Bio Whittaker Inc., Walkersville, Md.). Theintegrity of protein was verified by SDS-PAGE. Recombinant rat HMGB1(Wang et al., Science 285: 248-251, 1999) was used in some experimentssince it does not have degraded fragments as observed in purified humanHMGB1.

Peptide Synthesis

Peptides were synthesized and HPLC purified at Utah State UniversityBiotechnology Center (Logan, Utah) at 90% purity. Endotoxin was notdetectable in the synthetic peptide preparations as measured by Limulusassay.

Cell Culture

Murine macrophage-like RAW 264.7 cells (American Type CultureCollection, Rockville, Md.) were cultured in RPMI 1640 medium (LifeTechnologies, Grand Island N.Y.) supplemented with 10% fetal bovineserum (Gemini, Catabasas, Calif.), penicillin and streptomycin (LifeTechnologies) and were used at 90% confluence in serum-free Opti-MEM Imedium (Life Technologies, Grand Island, N.Y.). Polymyxin B (Sigma, St.Louis, Mo.) was routinely added at 100-1,000 units/ml to neutralize theactivity of any contaminating LPS as previously described; polymyxin Balone did not influence cell viability assessed with trypan blue (Wanget al., supra). Polymyxin B was not used in experiments of syntheticpeptide studies.

Measurement of TNF Release From Cells

TNF release was measured by a standard murine fibroblast L929 (ATCC,American Type Culture Collection, Rockville, Md.) cytotoxicity bioassay(Bianchi et al., Journal of Experimental Medicine 183:927-936, 1996)with the minimum detectable concentration of 30 pg/ml. Recombinant mouseTNF was obtained from R&D system Inc., (Minneapolis, MN). Murinefibroblast L929 cells (ATCC) were cultured in DMEM (Life Technologies,Grand Island, N.Y.) supplemented with 10% fetal bovine serum (Gemini,Catabasas, Calif.), penicillin (50 units/ml) and streptomycin (50 μg/ml)(Life Technologies) in a humidified incubator with 5% CO₂.

Antibody Production

Polyclonal antibodies against HMGB1 B box were raised in rabbits(Cocalico Biologicals, Inc., Reamstown, Pa.) and assayed for titer byimmunoblotting. IgG was purified from anti-HMGB1 antiserum using ProteinA agarose according to manufacturer's instructions (Pierce, Rockford,Ill.). Anti-HMGB1 B box antibodies were affinity purified by usingcyanogen bromide activated Sepharose beads (Cocalico Biological, Inc.).Non-immune rabbit IgG was purchased from Sigma (St. Louis, Mo.).Antibodies detected full length HMGB1 and B box in immunoassay, but didnot cross react with TNF, IL-1 and IL-6.

Animal Experiments

TNF knock out mice were obtained from Amgen (Thousand Oaks, Calif.) andwere on a B6x129 background. Age-matched wild-type B6x129 mice were usedas control for the studies. Mice were bred in-house at the University ofFlorida specific pathogen-free transgenic mouse facility (Gainesville,Fla.) and were used at 6-8 weeks of age.

Male 6-8 week old Balb/c and C3H/HeJ mice were purchased from HarlenSprague-Dawley (Indianapolis, Ind.) and were allowed to acclimate for 7days before use in experiments. All animals were housed in the NorthShore University Hospital Animal Facility under standard temperature,and a light and dark cycle.

D-galactosamine Sensitized Mice

The D-galactosamine-sensitized model has been described previously(Galanos et al., Proc Natl. Acad. Sci. USA 76: 5939-5943, 1979; andLehmann et al., J. Exp. Med. 165: 657663, 1997). Mice were injectedintraperitoneally with 20 mg D-galactosamine-HCL (Sigma)/mouse (in 200μl PBS) and 0.1 or 1 mg of either HMGB1 B box or vector protein (in 200μl PBS). Mortality was recorded daily for up to 72 hours afterinjection; survivors were followed for 2 weeks, and no later deaths fromB box toxicity were observed.

Statistical Analysis

Data are presented as mean±SEM unless otherwise stated. Differencesbetween groups were determined by two-tailed Student's t-test, one-wayANOVA followed by the least significant difference test or 2 tailedFisher's Exact Test.

EXAMPLE 2 Mapping the HMGB1 Domains for Promotion of Cytokine Activity

HMGB1 has 2 folded DNA binding domains (A and B boxes) and a negativelycharged acidic carboxyl tail. To elucidate the structural basis of HMGB1cytokine activity, and to map the inflammatory protein domain, fulllength and truncated forms of HMGB1 were expressed by mutagenesis andthe purified proteins were screened for stimulating activity in monocytecultures (FIG. 1). Full length HMGB1, a mutant in which the carboxyterminus was deleted, a mutant containing only the B box, and a mutantcontaining only the A box were generated. These mutants of human HMGB1were made by polymerase chain reaction (PCR) using specific primers asdescribed herein, and the mutant proteins were expressed using aglutathione S-transferase (GST) gene fusion system (Pharmacia Biotech,Piscataway, N.J.) in accordance with the manufacturer's instructions.Briefly, DNA fragments, made by PCR methods, were fused to GST fusionvectors and amplified in E. coli. The expressed HMGB1 protein and HMGB1mutants and were then isolated using GST affinity column.

The effect of the mutants on TNF release from Murine macrophage-like RAW264.7 cells (ATCC) was carried out as follows. RAW 264.7 cells werecultured in RPMI 1640 medium (Life Technologies, Grand Island N.Y.)supplemented with 10% fetal bovine serum (Gemini, Catabasas, Calif.),penicillin and streptomycin (Life Technologies). Polymyxin (Sigma, St.Louis, Mo.) was added at 100 units/ml to suppress the activity of anycontaminating LPS. Cells were incubated with 1 μg/ml of full length(wild-type) HMGB1 and each HMGB1 mutant protein in Opti-MEM I medium for8 hours, and conditioned supernatants (containing TNF which had beenreleased from the cells) were collected and TNF released from the cellswas measured by a standard murine fibroblast L929 (ATCC) cytotoxicitybioassay (Bianchi et al., supra) with the minimum detectableconcentration of 30 pg/ml. Recombinant mouse TNF was obtained from R & DSystems Inc., (Minneapolis, Minn.) and used as control in theseexperiments. The results of this study are shown in FIG. 1. Data in FIG.1 are all presented as mean+SEM unless otherwise indicated. (=6−10).

As shown in FIG. 1, wild-type HMGB1 and carboxyl-truncated HMGB1significantly stimulated TNF release by monocyte cultures (murinemacrophage-like RAW 264.7 cells). The B box was a potent activator ofmonocyte TNF release. This stimulating effect of the B box was specific,because A box only weakly activated TNF release.

EXAMPLE 3 HMGB1 B Box Protein Promotes Cytokine Activity in a DoseDependent Manner

To further examine the effect of HMGB1 B box on cytokine production,varying amounts of HMGB1 B box were evaluated for the effects on TNF,IL-1B, and IL-6 production in murine macrophage-like RAW 264.7 cells.RAW 264.7 cells were stimulated with B box protein at 0-10 μg/ml, asindicated in FIGS. 2A-2C for 8 hours. Conditioned media were harvestedand measured for TNF, IL-1β and IL-6 levels. TNF levels were measured asdescribed herein, and IL-1β and IL-6 levels were measured using themouse IL-1β and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (R&DSystem Inc., Minneapolis, 1) and N>5 for all experiments. The results ofthe studies are shown in FIGS. 2A-2C.

As shown in FIG. 2A, PNF release from RAW 264.7 cells increased withincreased amounts of B box administered to the cells. As shown in FIG.2B, addition of 1 μg/ml or 10 μg/ml of B box resulted in increasedrelease of IL-1β from RAW 264.7 cells. In addition, as shown in FIG. 2C,IL-6 release from RAW 264.7 cells increased with increased amounts of Bbox administered to the cells.

The kinetics of B box-induced TNF release was also examined. TNF releaseand TNF mRNA expression was measured in RAW 264.7 cells induced by B boxpolypeptide or GST tag polypeptide only used as a control (vector) (10μg/ml) for 0 to 48 hours. Supernatants were analyzed for TNF proteinlevels by an L929 cytotoxicity assay (N=3-5) as described herein. FormRNA measurement, cells were plated in 100 mm plate and treated inOpti-MEM I medium containing B box polypeptide or the vector alone for0, 4, 8, or 24 hours, as indicated in FIG. 2D. The vector only samplewas assayed at the 4 hour time point. Cells were scraped off the plateand total RNA was isolated by RNAzol B method in accordance with themanufacturer's instructions (Tel-Test “B”, Inc., Friendswood, Tex.). TNF(287 bp) was measured by RNase protection assay (Ambion, Austin, Tex.).Equal loading and the integrity of RNA was verified by ethidium bromidestaining of the RNA sample on agarose-formaldehyde gel. The results ofthe RNase protection assay are shown in FIG. 2D. As shown in FIG. 2D, Bbox activation of monocytes occurred at the level of gene transcription,because TNF mRNA was increased significantly in monocytes exposed to Bbox protein (FIG. 2B). TNF mRNA expression was maximal at 4 hours anddecreased at 8 and 24 hours. The vector only control (GST tag) showed noeffect on TNF mRNA expression. A similar study was carried out measuringTNF protein released from RAW 264.7 cells 0, 4, 8, 24, 32 or 48 hoursafter administration of B box or vector only (GST tag), using the L929cytotoxicity assay described herein. Compared to the control (mediumonly), B box treatment stimulated TNF protein expression (FIG. 2F) andvector alone (FIG. 2E) did not. Data are representative of threeseparate experiments. Together these data indicate that the HMGB1 B boxdomain has cytokine activity and is responsible for the cytokinestimulating activity of full length HMGB1.

In summary, the HMGB1 B box dose-dependently stimulated release of TNF,IL-1β and IL-6 from monocyte cultures (FIGS. 2A-2C), in agreement withthe inflammatory activity of full length HMGB1 (Andersson et al., J.Exp. Med. 192: 565-570, 2000). In addition, these studies indicate thatmaximum TNF protein release occurred within 8 hours (FIG. 2F). Thisdelayed pattern of TNF release is similar to TNF release induced byHMGB31 itself, and is significantly later than the kinetics of TNFinduced by LPS (Andersson et al., supra).

EXAMPLE 4 The First 20 Amino Acids of the HMGB1 B Box Stimulate TNFActivity

The TNF-stimulating activity of the HMGB1 B box was further mapped. Thisstudy was carried out as follows. Fragments of the B box were generatedusing synthetic peptide protection techniques, as described herein. FiveHMGB 1 B box fragments (from SEQ ID NO: 20), containing amino acids1-20, 16-25, 30-49, 45-64, or 60-74 of the HMGB1 B box were generated,as indicated in FIG. 3. RAW 264.7 cells were treated with B box (1μg/ml) or a synthetic peptide fragment of the B box (10 μg/ml), asindicated in FIG. 3 for 10 hours and TNF release in the supernatants wasmeasured as described herein. Data shown are mean±SEM, (n=3 experiments,each done in duplicate and validated using 3 separate lots of syntheticpeptides). As shown in FIG. 3, TNF-stimulating activity was retained bya synthetic peptide corresponding to amino acids 1-20 of the HMGB1 B boxof SEQ ID NO: 20 (fkdpnapkrlpsafflfcse; SEQ ID NO: 23). The TNFstimulating activity of the 1-20-mer was less potent than either thefull length synthetic B box (1-74-mer), or fall length HMGB1, but thestimulatory effects were specific because the synthetic 20-mers foramino acid fragments containing 16-25, 30-49, 45-64, or 60-74 of theHMGB 1 B box did not induce TNF release. These results are directevidence that the macrophage stimulating activity of the B boxspecifically maps to the first 20 amino acids of the HMGB B box domainof SEQ ID NO: 20). This B box fragment can be used in the same manner asa polypeptide encoding a full length B box polypeptide, for example, tostimulate releases of a proinflammatory cytokine, or to treat acondition in a patient characterized by activation of an inflammatorycytokine cascade.

EXAMPLE 5 HMGB1 B Box Protein is Toxic to D-galactosamine-sensitizedBalb/c Mice

To investigate whether the HMGB1 B box has cytokine activity in vivo, weadministered HMGB1 B box protein to unanesthetized Balb/c micesensitized with D-galactosamine (D-gal), a model that is widely used tostudy cytokine toxicity (Galanos et al., supra). Briefly, mice (20-25gram, male, Harlan Sprague-Dawley, Indianapolis, IN) wereintraperitoneally injected with D-gal (20 mg) (Sigma) and B box (0.1mg/ml/mouse or 1 mg/ml/mouse) or GST tag (vector; 0.1 mg/ml/mouse or 1mg/ml/mouse), as indicated in Table 1. Survival of the mice wasmonitored up to 7 days to ensure no late death occurred. The results ofthis study are shown in Table 1. TABLE 1 Toxicity of HMGB1 B box onD-galactosamine-sensitized Balb/c Mice Treatment Alive/total Control —10/10 Vector 0.1 mg/mouse 2/2   1 mg/mouse 3/3 B box 0.1 mg/mouse 6/6  1 mg/mouse  2/8*P < 0.01 versus vector alone as tested by Fisher's Exact Test

The results of this study showed that the HMGB1 B box was lethal toD-galactosamine-sensitized mice in a dose-dependent manner. In allinstances in which death occurred, it occurred within 12 hours.Lethality was not observed in mice treated with comparable preparationsof the purified GST vector protein devoid of B box.

EXAMPLE 6 Histology of D-Galactosamine-Sensitized Balb/c Mice or C3H/HeJMice Administered HMGB1 B Box Protein

To further assess the lethality of the HMGB1 B box protein in vivo theHMGB1 B box was again administered to D-galactosamine-sensitized Balb/cmice. Mice (3 per group) received D-gal (20 mg/mouse) plus B box orvector (1 mg/mouse) intraperitoneally for 7 hours and were thensacrificed by decapitation. Blood was collected, and organs (liver,heart, kidney and lung) were harvested and fixed in 10% formaldehyde.Tissue sections were prepared with hematoxylin and eosin staining forhistological evaluation (Criterion Inc., Vancouver, Canada). The resultsof these studies are shown in FIGS. 4A-4J, which are scanned images ofhematoxylin and eosin stained kidney sections (FIG. 4A), myocardiumsections (FIG. 4C), lung sections (FIG. 4E), and liver sections (FIGS.4G and 41 obtained from an untreated mouse and kidney sections (FIG.4B), myocardium sections (FIG. 4D), lung sections (FIG. 4F), and liversections (FIGS. 4H and 4J) obtained from mice treated with the HMGB1 Bbox. Compared to the control mice, B box treatment caused no abnormalityin kidneys (FIGS. 4A and 4B) and lungs (FIGS. 4E and 4F). The mice hadsome ischemic changes and loss of cross striation in myocardial fibersin the heart (FIGS. 4C and 4D as indicated by the arrow in FIG. 4D).Liver showed most of the damage by the B box as illustrated by activehepatitis (FIGS. 4G-4J). In FIG. 4J, hepatocyte dropouts are seensurrounded by accumulated polymorphonuclear leukocytes. The arrows inFIG. 4J point to the sites of polymorphonuclear accumulation (dotted) orapoptotic hepatocytes (solid). Administration of HMGB1 B box in vivoalso stimulated significantly increased serum levels of IL-6 (315+93 vs.20+7 pg/ml, B box vs. control, p<0.05) and IL-1β (15+3 vs. 4+1 pg/ml, Bbox vs. control, p<0.05).

Administration of B box protein to C3H/HeJ mice (which do not respond toendotoxin) was also lethal, indicating that HMGB1 B box is lethal in theabsence of LPS signal transduction. Hematoxylin and eosin stainedsections of lung and kidney collected 8 hours after administration of Bbox revealed no abnormal morphologic changes. Examination of sectionsfrom the heart however, revealed evidence of ischemia with loss of crossstriation associated with amorphous pink cytoplasm in myocardial fibers.Sections from liver showed mild acute inflammatory responses, with somehepatocyte dropout and apoptosis, and occasional polymorphonuclearleukocytes. These specific pathological changes were comparable to thoseobserved after administration of full length HMGB1 and confirm that theB box alone can recapitulate the lethal pathological response to HMGB1in vivo.

To address whether the TNF-stimulating activity of HMGB1 contributes tothe mediation of lethality by B box, we measured lethality in TNFknock-out mice (TNF-KO, Nowak et al., Am. J. Physiol. Regul. Integr.Comp. Physiol. 278: R1202-R1209, 2000) and the wild-type controls(B6x129 strain) sensitized with D-galactosamine (20 mg/mouse) andexposed to B box (1 mg/mouse, injected intraperitoneally). The B box washighly lethal to the wild-type mice (6 dead out of nine exposed) butlethality was not observed in the TNF-KO mice treated with B box (0 deadout of 9 exposed, p<0.05 v. wild type). Together with the data from theRAW 264.7 macrophage cultures, described herein, these data now indicatethat the B box of HMGB1 confers specific TNF-stimulating cytokineactivity.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A pharmaceutical composition comprising a polypeptide comprising anHMGB B box or a functional variant thereof, in an amount sufficient totreat a disease or condition by increasing an immune response in anindividual administered said pharmaceutical composition.
 2. Thepharmaceutical composition of claim 1, wherein said HMGB B box ismammalian.
 3. The pharmaceutical composition of claim 2, wherein saidHMGB B box is human.
 4. The pharmaceutical composition of claim 3,wherein said polypeptide comprises an HGB1 B box polypeptide.
 5. Thepharmaceutical composition of claim 4, wherein said polypeptide consistsof an HMGB1 B box polypeptide.
 6. The pharmaceutical composition ofclaim 1, further comprising a vaccine.
 7. The pharmaceutical compositionof claim 6, further comprising an adjuvant.
 8. The pharmaceuticalcomposition of claim 7, wherein said adjuvant is selected from the groupconsisting of one or more immunostimulatory oligonucleotides, animidazoquinoline, monophosphoryl lipid A, Brad detoxifiedlipopolysaccharide.
 9. The pharmaceutical composition of claim 8,wherein said immunostimulatory oligonucleotides comprise unmethylatedCpG sequences.
 10. An antibody attached to a polypeptide comprising anHMGB B box or a functional variant there of.
 11. The antibody of claim10, wherein said HMGB B box is mammalian.
 12. The antibody of claim 11,wherein said HMGB B box is human.
 13. The antibody of claim 12, whereinsaid polypeptide comprises an HMGB1 B box polypeptide.
 14. The antibodyof claim 13, wherein said polypeptide consists of an HMGB1 B boxpolypeptide.
 15. The antibody of claim 10, wherein said antibody binds arumor-associated polypeptide.
 16. The antibody of claim 10, wherein saidantibody is in a pharmaceutically acceptable carrier.
 17. A method ofsimulating or increasing an immune response in an individual in need ofimmunostimulation, said method comprising administering to saidindividual a polypeptide comprising an HMGB B box or a functionalvariant thereof, in a amount sufficient to stimulate or increase saidimmune response.
 18. The method of claim 17, wherein said individual isbeing treated for cancer.
 19. The method of claim 17, wherein said HMGBB box is mammalian.
 20. The method of claim 19, wherein said HMGB B boxis human.
 21. The method of claim 20, wherein said polypeptide comprisesan HMGB1 B box.
 22. The method of claim 21, wherein said polypeptideconsists of an HMGB1 B box.
 23. The method of claim 17, wherein saidpolypeptide is co-administered with a vaccine.
 24. The method of claim23, wherein said polypeptide is co-administered with a m furtheradjuvant.
 25. The method of claim 24, wherein said adjuvant is selectedfrom the group consisting of one or more immunostimulatoryoligoneucleotides, an imidazoquinoline, monophosphoryl lipid A, anddetoxified W lipopolysaccharide.
 26. The method of claim 25, whereinsaid immunostimulatory oligonucleotides comprise unmethylated CpGsequences.
 27. The method of claim 17, wherein said administration issystemic.
 28. The method of claim 17, wherein said administration islocalized to a target site.
 29. The method of claim 17, wherein saidpolypeptide is attached to an antibody specific to a target site in theindividual in need of immunostimulation.
 30. The method of claim 17,wherein said polypeptide is in a pharmaceutically acceptable carrier.31. A method of treating cancer in an individual, said method comprisingadministering to said individual a therapeutically effective amount of apolypeptide comprising an HMGB B box or a functional variant thereof.32. The method of claim 31, wherein said HMGB B box is mammalian. 33.The method of claim 32, wherein said HMGB B box is human.
 34. The methodof claim 33, wherein said polypeptide comprises an HMGB1 B boxpolypeptide.
 35. The method of claim 34, wherein said polypeptideconsists of an HMGB1 B box polypeptide.
 36. The method of claim 31,wherein said polypeptide is co-administered with a vaccine.
 37. Themethod of claim 36, wherein said polypeptide is co-administered with afurther adjuvant.
 38. The method of claim 37, wherein said adjuvant isselected from the group consisting of one or more immunostimulatoryoligonucleotides, an imidazoquinoline monophosphoryl lipid A, anddetoxified lipopolysacharide.
 39. The method of claim 38, wherein saidimmunostimulatory oligonucleotides comprise unmethylated CpG sequences.40. The method of claim 31, wherein said administration is systemic. 41.The method of claim 31, wherein said administration is localized to atarget site.
 42. The method of claim 41, wherein said target site is atumor.
 43. The method of claim 31, wherein said polypeptide is attachedto an antibody.
 44. The method of claim 43, wherein said antibody bindsa tumor-associated polypeptide.
 45. The method of claim 31, wherein saidpolypeptide is in a pharmaceutically acceptable carrier.